Few foreign hemoproteins have been expressed in bacteria. Expression of active sperm whale [Springer and Sligar, Proc. Natl. Acad. Sci. USA 84: 8961-8965(1987)] and human [Varadarajan et al., Proc. Natl. Acad. Sci. 82: 5681-5684 (1985)] myoglobins, rat heme oxygenase [Ishikawa et al., Eur. J. Biochem. 303: 161-163 (1991)] and horseradish peroxidase C [Smith et al., J. Biol Chem. 265:13335-13343 (1990)] in E. coli have been reported. The myoglobins were purified to homogeneity from the crude bacterial extracts. Heme was either taken from the host bacteria [Varadarajan et al., Proc. Natl. Acad. Sci. 82: 5681-5684 (1985)] or incorporated into the apoprotein in vitro after the purification [Springer and Sligar, Proc. Natl. Acad. Sci. USA 84: 8961-8965(1987)]. Heme oxygenase, on the other hand, remained bound to the bacterial membranes and its heme saturation was uncertain [Ishikawa et al., Eur. J. Biochem. 303:161-163 (1991)]. Horseradish peroxidase C is produced in inclusion bodies in an insoluble inactive form containing only traces of heme and requires a tedious solubilization and in vitro reactivation [Smith et al., J. Biol Chem. 265:13335-13343 (1990)]. These examples demonstrate that expression of a hemoprotein in bacteria in an active, soluble form sufficiently saturated with heme often represents a difficult task.
It has been demonstrated recently that cystathionine .beta.-synthase [L-serine hydrolyase (adding homocysteine); E. C. 4.2.1.22; CBS] is a hemoprotein [Bukovska et al., Protein Express. Purif. 5: 442-448 (1994); Kery et al., J. Biol. Chem. 269: 25283-25288 (1994)]. CBS is central to sulfur amino acid and S-adenosyl-L-methionine (AdoMet) metabolism in eukaryotes [Mudd et al., The Metabolic Basis of Inherited Disease 6th ed., pp 693-734, McGraw-Hill, New York (1989)]. It catalyzes the condensation of serine and homocysteine to cystathionine, the first step of the pyridoxal 5'-phosphate dependent (PLP) transsulfuration to cysteine. cDNAs encoding rat [Swaroop et al., J. Biol. Chem. 267: 11455-11461 (1992)] and human [Kraus et al., Human Mol. Genet. 2: 1633-1638 (1993)] CBS have been cloned and their amino acid sequences have been deduced. The human CBS cDNA was cloned into the pAX5.sup.- plasmid and expressed in Escherichia coli XL1 Blue MR as a fusion protein with .beta.-galactosidase. The fusion protein was cleaved by a specific protease and the released CBS was purified to homogeneity [Bukovska et al., Protein Express. Purif. 5: 442-448 (1994)].
Basic characteristics of the cloned human CBS were nearly identical to those determined previously for the rat and human liver enzyme [Bukovska et al., Protein Express. Purif. 5: 442-448 (1994)]: The enzyme required heme in addition to PLP for its function and was activated by AdoMet. Heme had to be present at the time of protein folding to be properly incorporated into the enzyme [Kery et al., J. Biol. Chem. 269: 25283-25288 (1994)].
Routine purification of the cloned human CBS yields enzyme which is an average only 20% saturated with heme. This low heme saturation substantially decreases the production of an active enzyme in E. coli because no in vitro method has been found which would allow reconstitution of the unsaturated enzyme with heme [Kery et al., J. Biol. Chem. 269: 25283-25288 (1994)].